Background: c-Cbl associates with various signaling molecules to regulate diverse signaling networks via ubiquitination of receptors and/or protein tyrosine kinases. Results: c-Cbl drives ubiquitin-dependent Ag⅐BCR trafficking to MIIC and BCR-mediated antigen processing and presentation. Conclusion: c-Cbl directs BCR-mediated antigen processing and presentation. Significance: c-Cbl coordinates antigen-induced BCR signaling and rapid BCR-mediated antigen processing and presentation to drive a strong humoral immune response.
The gram-negative bacterium Francisella tularensis (Ft) is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize Ft grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to Ft grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select Ft virulence factors by an unknown mechanism. Here, we tested the hypotheses that Ft grown in BHI (BHI-Ft) accurately displays a full protein composition more similar to that reported for infection-derived Ft and that this similarity would make BHI-Ft more susceptible to pre-existing, vaccine-induced immunity than MHB-Ft. We performed comprehensive proteomic analysis of Ft grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published “omics” data derived from Ft grown in vivo. Based on the abundance of ~1,000 proteins, the fingerprint of BHI-Ft is one of nutrient-deprived bacteria that—through induction of a stringent-starvation-like response—have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI-Ft would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown Ft S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent.
B cell receptor (BCR)-mediated antigen processing and presentation is critical to the initiation and control of a humoral immune response. Trafficking of internalized antigen-BCR (Ag-BCR) complexes to intracellular antigen processing compartments is driven by ubiquitination of the cytoplasmic domain of the BCR. Using a biochemical approach, it is here established that ubiquitinated Ag-BCR complexes are formed via a signaling-dependent mechanism and restricted to plasma membrane lipid rafts. Since the structure of lipid rafts is temperature sensitive, the impact of physiological range temperature changes (33–39°C) on lipid raft-dependent and independent BCR functions was investigated. While the kinetics of lipid raft independent BCR internalization are unaffected by temperature changes within this range, raft-dependent BCR signaling and ubiquitination as well as BCR-mediated antigen processing are significantly impacted. When compared to 33°C (peripheral body temperature), the extent and duration of Ag-BCR ubiquitination is increased and prolonged at 37–39°C (normal to febrile temperature). As might be expected, increased temperature also accelerates the overall kinetics of Ag-BCR degradation. Interestingly, at 33°C the expression of peptide-class II complexes derived from the BCR-mediated processing of cognate antigen is profoundly slowed, whereas the kinetics of expression of peptide-class II complexes derived from fluid-phase antigen processing remain unchanged. These results establish the effect of physiological range temperature changes on multiple lipid raft-dependent BCR functions including the processing and presentation of cognate antigen, suggesting one mechanism by which physiological range temperature changes such as fever may impact the initiation and /or maturation of a humoral immune response.
Francisella tularensis (Ft) is a biothreat agent for which there is no FDA-approved human vaccine. Currently, there are substantial efforts underway to develop both vaccines and the tools to assess these vaccines. Tularemia laboratory research has historically relied primarily upon a small number of inbred mouse strains, but the utility of such findings to outbred animals may be limited. Specifically, C57BL/6 mice are more susceptible than BALB/c mice to Ft infection and less easily protected against challenge with highly virulent type A Ft. Thus, depending on the inbred mouse strain used, one could be misled as to which immunogen(s)/vaccine will ultimately be effective in an outbred human population. Accordingly, we evaluated an outbred Swiss Webster (SW) mouse model in direct comparison to a well-established, inbred C57BL/6 mouse model. Mucosal vaccination with the live, attenuated Ft LVS superoxide dismutase (sodB) mutant demonstrated significantly higher protection in outbred SW mice compared to inbred C57BL/6 mice against Ft SchuS4 respiratory challenge. The protection observed in vaccinated outbred mice correlated with lower bacterial density, reduced tissue inflammation, and reduced levels of pro-inflammatory cytokine production. This protection was CD4+ and CD8+ T cell-dependent and characterized by lower titers of serum antibody (Ab) that qualitatively differed from vaccinated inbred mice. Enhanced protection of vaccinated outbred mice correlated with early and robust production of IFN-γ and IL-17A. Neutralizing Ab administered at the time of challenge revealed that IFN-γ was central to this protection, while IL-17A neutralization did not alter bacterial burden or survival. The present study demonstrates the utility of the outbred mouse as an alternative vaccination model for testing tularemia vaccines. Given the limited MHC repertoire in inbred mice, this outbred model is more analogous to the human in terms of immunological diversity.
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